Cell surface heparan sulfate (HS) interacts with diverse proteins including the microbial surface proteins involved in attachment and/or entry into the host. The ability of HS to act as an attachment as well as membrane fusion receptor for herpes simplex virus (HSV) relates to its structural and functional diversity originating from extensive modifications during its biosynthesis. The final modification of HS is the 3-O sulfation which is mediated by 3-O-sulfotransferases (3-OSTs). Seven members of the 3-OST family have been identified and it has been suggested that each of the 3-OSTs are capable of recognizing unique saccharide sequences around the modification sites. This site-specific function of each isoform allows them to generate their own distinct 3-O-sulfated heparan sulfate (3-OS HS) motifs. In order to identify small peptide inhibitors of HS and 3-OS HS functions especially during HSV-1 entry, we screened both unmodified HS and 3-OS HS generated by 3-O-sulfotransferase-3 (3-OST-3) using 12 mer-phage display library and isolated a panel of peptides with unique sequence diversity. Of which, we characterized the G1 and G2 peptides isolated against unmodified HS and modified HS respectively. The characterization of G1 and G2 peptides demonstrated promising results by inhibiting HSV-1 infections both in vitro and in vivo models of ocular HSV-1 infection and genital herpes infection. However, several of the anti-HS and anti-3O HS peptides identified during initial screening have not been tested against HSV-1 infection. Our hypothesis is that characterization of the entire panel of anti-HS and anti-3-OS HS peptide will not only increase our chance to isolate the most potent inhibitor against HS and modified HS respectively but it will also lead towards the development of novel reagents to map out structurally complex chains of HS. Aim 1 of the proposal will focus on synthesis and functional characterization of anti-3-O-sulfated heparan sulfate (3-OS HS) binding peptides against HSV-1 entry and cell-to-cell spread. We will generate detailed information on the abilities of the individual peptides to interfere with HSV-1 attachment, entry and cell-to-cell spread. Aim 2 will determine the anti-HSV potential of 3- OS HS peptides against multiple 3-OST isoforms. It will also determine the relative cross specificities of the peptides against the 3 OS HS generated by other isoforms. Our study will produce some valuable reagents against various 3-OS HS modifications and help greatly with the understanding of HS functions in human biology and infectious diseases.